Assessment of protocols and variance for specific leaf area (SLA) in 10 Eucalyptus species to inform functional trait sampling strategies for TERN Ausplots

IntroductionFunctional trait-based approaches to ecological questions are becoming more common. The ongoing development of large functional trait databases has further enabled these kinds of studies. TERN is Australia's national land ecosystem observatory. Through monitoring more than 750 plots across major biomes and bioclimatic gradients, TERN Ecosystem Surveillance has collected over 40,000 voucher specimens with herbarium level taxonomic determinations. This collection represents an opportunity to generate high quality functional trait data for a large number of Australian flora.This pilot study aimed to test the feasibility of using the TERN collection to measure a simple morphological trait. Specific leaf area (SLA) is the one-sided area of a fresh leaf divided by its dry mass. We restricted our study to the Eucalyptus genus as Eucalyptus species are common in TERN monitoring plots and are often the dominant tree species. The results of the study should inform the future sampling strategy for SLA.MethodThe first component of the study was the measurement of leaves from voucher specimens. We located 30 Eucalyptus vouchers exclusively from South Australian plots (figure 1) and took 5 leaves from each specimen. The leaves were mounted onto sheets of paper and scanned with a flatbed scanner. Leaf area was measured from the scans using ImageJ software. The mounted leaves were placed into a plant press and dried in an oven for 24 hours at 70C. Each leaf was individually weighed using a 0.1mg microbalance.The second component was the collection and measurement of fresh leaf samples. We collected 5 leaves from 5 individuals for 3 species growing at Waite Conservation Reserve in Adelaide, South Australia. The leaves were mounted, scanned and measured as before. They were then dried in an oven for 72 hours at 70C and weighed with the same microbalance. The dried leaves were scanned and measured again so that leaf area shrinkage between fresh and dry leaves could be estimated. Shrinkage percentage was obtained using the formula: ((fresh area - dry area) / fresh area) * 100SLA was obtained for each leaf using the formula: dry area / dry massWe ran an Anova (type II) on our SLA data using the Car v3.0-10 package in R v4.0.3.ResultsThe pilot dataset contained 225 leaves from 45 individuals encompassing 10 species.The mean shrinkage in leaf area was 10.27% with a standard deviation of 1.75%. This estimate came from 75 leaves (25 each from E. microcarpa, E. leucoxylon and E. camaldulensis subsp. camaldulensis).The Anova output (figure 2) revealed that variation between individuals of the same species contributed the most to the overall SLA variation (sumsq = 111.6, R^2 = 0.499). A substantial portion of the overall variation was also attributed to variation between species (sumsq = 88.5, R^2 = 0.396%). The residual variation (sumsq = 23.3, R^2 = 0.104) was attributed to the variation between leaves from the same individual. The boxplots of 'SLA by individual' and 'SLA by species' (figure 3) support these results.RecommendationsThe shrinkage results show that shrinkage due to water loss is consistent and predictable for Eucalyptus leaves. This means that leaf traits can be reliably measured from herbarium style collections and the data derived from such measurements can be compared and integrated with data from fresh leaves.By analysing the variance in the SLA data we have shown that the variation between individuals of the same species is significant and deserves further attention. However, the variation between species is also significant and should be captured in future studies. As such, we recommend that any subsequent attempt to construct a larger dataset of SLA measurements from the TERN voucher collection should focus on Eucalyptus species which are well represented. This will ensure that both intraspecific and interspecific variation is captured. Currently there are 27 Eucalyptus species with 10 or more vouchers, 47 species with 5 or more vouchers and 130 species with 1 or more voucher. The variation between individual leaves was found to be a small part of the overall variation. This means that in future it should not be necessary to measure 5 leaves from each individual. Measuring 1 leaf from an individual will likely give a reliable estimate of the individual's mean SLA.Certain changes to the TERN survey methodology could help to facilitate the accumulation of trait data. It is important that plant material taken for vouchers is the youngest fully mature material available and that it is from the outermost part of the canopy (i.e. sun leaves). This will help to ensure consistency and accuracy of trait measurements. When fruit/seeds are present they should be placed into a bag and kept with the voucher specimen. Taking ample plant material from each individual will ensure that destructive trait analysis does not affect the quality of the voucher specimen. Where a species is abundant or dominant it will be beneficial to take vouchers from multiple individuals to further investigate intraspecific and within-site trait variation. This study has served to highlight the potential for a trait database to be produced from the TERN voucher collection. It is evident that, for at least the Eucalyptus genus, there is valuable trait information contained in specimen vouchers which, if measured, could enable further research into important ecological questions (e.g. how does SLA vary intraspecifically and does it contribute to a species' environmental tolerance?).ReferencesGarnier, E, Shipley, B, Roumet, C & Laurent, G 2001, 'A standardized protocol for the determination of specific leaf area and leaf dry matter content', Functional Ecology, vol. 15, pp. 688-695.Munroe, S, Guerin, GR, Saleeba, T, , Martin-Fores, M, Blanco-Martin, B, Sparrow, B & Tokmakoff, A 2020. 'ausplotsR: An R package for rapid extraction and analysis of vegetation and soil data collected by Australia’s Terrestrial Ecosystem Research Network'. EcoEvoRxiv, DOI 10.32942/osf.io/25phxNock, CA, Vogt, RJ & Beatrix, BE 2016, 'Functional Traits', eLS.Pérez-Harguindeguy, N, Díaz, S, Garnier, E, Lavorel, S, Poorter, H, Jaureguiberry, P, Bret-Harte, MS, Cornwell, WK, Craine, JM, Gurvich, DE, Urcelay, C, Veneklaas, EJ, Reich, PB, Poorter, L, Wright, IJ, Ray, P, Enrico, L, Pausas, JG, de Vos, AC, Buchmann, N, Funes, G, Quétier, F, Hodgson, JG, Thompson, K, Morgan, HD, ter Steege, H, van der Heijden, MGA, Sack, L, Blonder, B, Poschlod, P, Vaieretti, MV, Conti, G, Staver, AC, Aquino, S & Cornelissen, JHC 2016, 'New handbook for standardised measurement of plant functional traits worldwide', Australian Joural of Botany, vol. 64, pp. 715-716.Perez, TM, Rodriguez, J & Heberling, JM 2020, 'Herbarium-based measurements reliably estimate three functional traits', American Journal of Botany, vol. 107, no. 10, pp. 1457-1464.Queenborough, S 2017, 'Collections-based Studies of Plant Functional Traits', Scientia Danica, Series B, no. 6, pp. 223-236.Sparrow, B, Foulkes, J, Wardle, G. Leitch, E, Caddy-Retalic, S, van Leeuwen, S, Tokmakoff, A, Thurgate, N, Guerin, G & Lowe, A 2020. 'A Vegetation and soil survey method for surveillance monitoring of rangeland environments'. Frontiers in Ecology and Evolution, vol. 8, pp. 157.Tavsanoglu, C & Pausas, JG 2018, 'A functional trait database for Mediterranean Basin plants', Scientific Data, vol. 5, pp. 1-18.Torrez, V, Jorgensen, PM & Zanne, AE 2012, 'Specific leaf area: a predictive model using dried samples', Australian Journal of Botany, vol. 61, pp. 350-357.

生态学领域基于功能性状的探究方法正日益普及。大型功能性状数据库的持续发展进一步推动了此类研究的发展。TERN（澳大利亚国家陆地生态系统观测站）通过监测主要生物群落和生物气候梯度中的750多个样地，收集了超过40,000份具有标本馆水平分类鉴定的凭证标本。这一收藏为大量澳大利亚植物生成高质量功能性状数据提供了机遇。本试点研究旨在验证利用TERN收藏测量简单形态性状的可行性。特定叶面积（SLA）是指新鲜叶片单侧面积与其干重之比。本研究将范围限定于桉属，因为桉树在TERN监测样地中普遍存在，且常常是优势树种。研究结果应能为SLA的未来采样策略提供信息。方法：研究的第一个组成部分是对凭证标本中叶片的测量。我们从南澳大利亚的样地中定位了30份桉树凭证标本（图1），并从每个标本中取出5片叶子。叶片被固定在纸上并使用平板扫描仪进行扫描。使用ImageJ软件从扫描图像中测量叶片面积。固定后的叶片被放置在植物压片中，并在70℃的烤箱中干燥24小时。每个叶片单独使用0.1mg微天平称重。第二个组成部分是收集和测量新鲜叶片样本。我们从南澳大利亚阿德莱德的Waite自然保护区3个物种的5个个体中各收集了5片叶子。叶片如前所述进行固定、扫描和测量。然后，它们在70℃的烤箱中干燥72小时，并使用相同的微天平称重。干燥后的叶片再次进行扫描和测量，以估算新鲜叶片与干燥叶片之间的面积收缩率。使用以下公式计算收缩百分比：((新鲜面积 - 干燥面积) / 新鲜面积) * 100。使用以下公式计算每个叶片的SLA：干燥面积 / 干重。我们使用R v4.0.3中的Car v3.0-10包对SLA数据进行Anova（第二类）分析。结果：试点数据集包含来自45个个体、涵盖10个物种的225片叶子。叶片面积的收缩平均值是10.27%，标准差为1.75%。这一估计来自75片叶子（每个物种25片，分别为E. microcarpa、E. leucoxylon和E. camaldulensis subsp. camaldulensis）。Anova输出结果（图2）显示，同一物种个体间的差异对SLA总变异的贡献最大（sumsq = 111.6，R^2 = 0.499）。SLA总变异的相当一部分也归因于物种间的差异（sumsq = 88.5，R^2 = 0.396%）。残差变异（sumsq = 23.3，R^2 = 0.104%）归因于同一个体叶片间的差异。'个体SLA'和'物种SLA'的箱线图（图3）支持这些结果。建议：收缩结果表明，由于水分损失引起的收缩对于桉树叶来说是稳定且可预测的。这意味着可以从标本馆风格的收藏中可靠地测量叶片性状，并且从这些测量中获得的数据可以与新鲜叶片的数据进行比较和整合。通过分析SLA数据的变异性，我们已表明同一物种个体间的差异是显著的，并值得进一步关注。然而，物种间的差异也是显著的，应在未来的研究中得到捕捉。因此，我们建议任何后续尝试从TERN凭证收藏构建更大SLA测量数据集的努力应集中在桉树物种上，这些物种在凭证收藏中得到了很好的代表。这将确保捕捉到种内和种间变异。目前有27个桉树物种有10份或更多的凭证，47个物种有5份或更多的凭证，130个物种有1份或更多的凭证。个体叶片间的差异被发现是总体变异的一小部分。这意味着在未来，不需要从每个个体测量5片叶子。测量一个个体的1片叶子可能就能提供一个可靠的SLA平均值。对TERN调查方法的某些调整有助于促进性状数据的积累。重要的是，用于凭证的植物材料是可获得的最新完全成熟材料，并且来自树冠的最外侧部分（即阳光叶子）。这将有助于确保性状测量的连贯性和准确性。当存在果实/种子时，它们应放入袋子中，并与凭证标本一起保存。从每个个体采集足够的植物材料将确保破坏性性状分析不会影响凭证标本的质量。当一个物种丰富或占主导地位时，从多个个体采集凭证将有助于进一步研究种内和局部场所性状变异。本研究旨在凸显从TERN凭证收藏中产生性状数据库的潜力。对于至少桉属而言，标本凭证中包含有价值的性状信息，如果进行测量，将能够促进对重要生态问题的进一步研究（例如，SLA在种内如何变化，它是否有助于物种的环境耐受性？）。参考文献：Garnier, E, Shipley, B, Roumet, C & Laurent, G 2001, 'A standardized protocol for the determination of specific leaf area and leaf dry matter content', Functional Ecology, vol. 15, pp. 688-695。